Astrocytes have characteristic intramembranous particle assemblies. Their distribution is polarized in vivo, with the highest concentration in the astroglial membranes facing blood vessels, pia and the lowest satellite to neurons. To test whether the opposing blood vessels, pia and neurons affect the assembly number, astrocytes from neonate rats were cultured alone or with different cell types and examined with freeze-fracture electron microscopy. Co-culturing astrocytes with brain endothelium, meningeal cells or fibroblasts consistently increased the assembly number 3-6 fold over a 6-17 day period. Bovine pulmonary artery, aortic endothelium and a tracheal- derived cell line were ineffective. The effect of CNS neurons could not be assessed because they could not be separated from their intrinsic astrocytes. Agents that elevate the activity of cyclic adenosine phosphate (cAMP), augmented the assembly number most dramatically. A single pulse of forskolin, which raises cAMP levels in astroglia, increased the assembly number 15 fold after 3 days. The beta agonist, isoproterenol, which also raises cAMP levels in astrocytes, also increased assemblies, especially when 3-isobutyl-l-methyl-xanthine, which inhibits cAMP degradation, was included. These results suggest that cAMP may be linked to the presence of assemblies in the astroglial cell membrane. During mitogen stimulation of C6 glioma and a human cell line of glial cells in vitro, the typical isomers of phosphatylinositol (PI) degradation were formed. These products included glycerol phosphate inositol diphosphate and glycerol phosphate inositol phosphate, which are rarely found in non-glial cells. In C6 cells, alpha-adrenergic and serotonergic stimulation increased the amount of the PI intermediates. Astroglial proliferation is accompanied by stimulation of the PI cycle.